Tracking single particles for hours via continuous DNA-mediated fluorophore exchange

Abstract

Monitoring biomolecules in single-particle tracking experiments is typically achieved by employing fixed organic dyes or fluorescent fusion proteins linked to a target of interest. However, photobleaching typically limits observation times to merely a few seconds, restricting downstream statistical analysis and observation of rare biological events. Here, we overcome this inherent limitation via continuous fluorophore exchange using DNA-PAINT, where fluorescently-labeled oligonucleotides reversibly bind to a single-stranded DNA handle attached to the target molecule. Such versatile and facile labeling allows uninterrupted monitoring of single molecules for extended durations. We demonstrate the power of our approach by observing DNA origami on membranes for tens of minutes, providing perspectives for investigating cellular processes on physiologically relevant timescales.

Design Features to Accelerate the Higher-Order Assembly of DNA Origami on Membranes

“…To study DNA origami cross-linking, we first designed a suitable monomer structure. We reasoned that the use of a well-characterized modular structure would be most convenient and thus opted for a flat rectangular grid origami used in a number of previous single-molecule fluorescence studies. ,,,, On this monomer structure, we arranged 36 DNA-PAINT docking sites in the shape of an arrow. This design challenges the resolution in DNA-PAINT imaging and allows reading out the orientation of the origami on the surface (Figure ).…”

“…The other strategy is to incorporate modified staples into the DNA origami that carry extensions for indirect binding of connector strands to the DNA origami. We reasoned that DNA origami superstructure assembly could be accelerated through a connector strand design analogous to the above-mentioned high-on-rate docking site design ,, used for example in DNA-PAINT, i.e., the use of low-complexity sequences to increase the effective association rate (Figure c). We opted for short stretches of a single nucleotide species, specifically A 7 as an extreme case of such a low-complexity sequence.…”

“…DNA origami with a 20 nucleotide (nt) adapter sequence were deposited on membranes. A [TCT] 38 “tracking handle” docking site analogous to that described by Stehr et al 29 was quasi-irreversibly recruited to the origami via the adapter complement: During DNA origami deposition, 10 nM tracking-handle–adapter conjugate were additionally present. To ensure a sparse subset of labeled DNA origami nanostructures suitable for SPT, a low density of tracking-handle-coupled particles was diluted in a 20-fold excess of unlabeled DNA origami particles, i.e., the same DNA origami, except without the adapter sequence.…”

“… 28 In this case, a long docking strand and a high concentration of imager strands yield unusually long tracks due to continuous replacement of bleached imager strands, circumventing photobleaching limitations to track duration. 29 …”

“…The same strategy can also be used in single-particle tracking (SPT) of sparse sets of DNA origami particles . In this case, a long docking strand and a high concentration of imager strands yield unusually long tracks due to continuous replacement of bleached imager strands, circumventing photobleaching limitations to track duration …”